Prenylation of proteins is a relatively newly discovered post-translational modification of proteins. Although the phenomenon has been intensively studied for a number of years, the properties imparted by this modification remain obscure in most cases. Prenylation has been implicated in membrane binding, protein-protein interactions and conformation changes. This proposal seeks to address several specific questions involving the effects of prenylation upon the interaction of the small GTP-binding protein RhoA with one of its regulatory proteins, Rho-GDI (Rho-Guanosine Nucleotide Dissociation Inhibitor). Rho-GDI is the major modulator of Rho activity and hence understanding the interaction of these two proteins is of primary importance in understanding the function of Rho. The main thrust of these studies is to determine how alternative C-terminal post-translational processing of Rho affects the binding of Rho to Rho-GDI. We will attempt to determine the dependence of the binding of these two proteins upon the pattern of prenylation (i.e. the Ras motif versus. the Rho motif) and the structure of the prenyl group. We will also attempt to identify regions of the protein, remote from the prenylated carboxy-terminus of RhoA, that are important for the interaction of RhoA with Rho-GDI. Finally, we will attempt to unequivocally establish the existence of a direct prenyl group-regulatory protein interaction through the use of a Rho protein modified with a photoactive analog of the normal prenyl group. While widely assembled to be an important component of the interaction Rho with Rho-GDI, a direct interaction between the prenyl group and any regulatory protein has yet to be demonstrated explicitly.